Abstract MicroRNAs (miRNAs) are a recently discovered class of small, ~22-nt regulatory RNAs that are now known to play key roles in the regulation of cellular differentiation and development. Misregulation of miRNA expression can contribute to disease progression, particularly in the case of cancer, and recent evidence also implicates miRNAs in aspects of viral replication and pathogenesis. Several viruses, including numerous herpesviruses and polyomavirus species, are now known to express a range of virally-encoded miRNAs, and virus infection is also known to perturb cellular miRNA expression in ways that may facilitate virus replication. Although relatively little is know about how HIV-1 interacts with the cellular miRNA machinery, it appears clear that HIV-1 infection can modify the pattern of cellular miRNA expression, and individual cellular miRNAs have been proposed to either facilitate or inhibit HIV-1 replication. It also remains possible that HIV-1 may encode one or more miRNAs, although this has been controversial. In this grant application, we propose to systematically analyze the effect of HIV-1 infection on the pattern of miRNA expression in primary CD4+ T cells and macrophages using microarray and deep sequencing technologies. We will then use cross-linking immunoprecipitation (CLIP) technologies to identify all the RNA induced silencing complex (RISC) binding sites on all the mRNAs expressed in HIV-1-infected cells by using an Argonaute-specific monoclonal antibody to recover cross-linked mRNA:RISC complexes, which we will then analyze by deep sequencing. Cellular or HIV- 1 mRNAs that are targeted by miRNAs expressed in HIV-1-infected cells will then be subjected to mutational analysis, combined with functional assays, to identify mRNA:miRNA interactions that modulate the efficiency of HIV-1 replication. Finally, we will determine whether drug-specifically cocaine-abuse modifies the miRNA expression profile in uninfected and HIV-1-infected CD4+ T cells and macrophages and we will examine whether any drug-induced changes in the miRNA profile can account for the reported enhancement in HIV-1 replication in cells isolated from drug-abusing patients. Together, this analysis will lead to a comprehensive understanding of how HIV-1 infection modifies the miRNA expression profile and will provide a mechanistic understanding of how these changes regulate the efficiency of HIV-1 replication.